The overall goals of this proposal are to introduce, by design, a series of binding sites for redox active metal centers near the pre-exiting heme co-factor of an oxidative heme enzyme in order to study the electron-transfer, electronic coupling, and redox reactions that result from the interaction of the two metal centers. Such binuclear metalloprotein complexes may serve as mechanistic and functional models of naturally occurring enzymes such as manganese dependent ligninase and cytochrome c oxidase. Metal binding sites have been introduced into cytochrome c peroxidase near the heme periphery that result in significantly enhanced enzyme dependent oxidation of Mn+2 ions by H2O2. Continued studies will provide insights in two distinctly different arenas: 1) By comparing the kinetics, spectroscopy, energetics and co- factor dependence of these hybrids, significant insight may be obtain into the relative importance of parameters such as ligand environment, metal-heme coupling, and redox thermodynamics. Thus, these studies may provide answers to unresolved questions about the function of natural systems containing interacting heme and metal centers. 2) The accumulation of new information on the binding specificity, thermodynamics and structural parameters of several related designed metal binding sites in the context of a well characterized model protein framework containing a second redox active co-factor will provide important input for the overall design goals of the metalloprotein design program. The experiments in project VII are meant to complement work on other designed metalloproteins, yet focus upon details of metal- co-factor interactions. In addition, we will perform supporting and collaborative studies within the program project to help characterize designed metal sites near the chromophore of green fluorescent protein (GFP). EPR, electrochemistry, calorimetry and fluorescence kinetics experiments intended to explore the effects of metal binding on the properties of the chromophore will provide significant feedback on these project components. In this way, the proposed studies will directly impact on the central themes of the program project by contributing to a rule base for the design and evaluation of metal sites within protein structures for the purpose of controlling the regulation, folding and function of novel metalloproteins.